A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect.

Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines). Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect. Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer (read more)

Flow Cytometry Crossmatch Reactivity With Pronase-Treated T Cells Induced by Non-HLA Autoantibodies in Human Immunodeficiency Virus-Infected Patients.

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However, we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study, 25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not have any effect on the T cell FCXM reactivity. Pretreatment with reducing agents did not affect the T cell FCXM reactivity. 15 of 21 HIV-infected kidney allograft recipients with pronase-treated T cell FCXM reactivity display long-term graft survival (1,193 ± 631 days). These data indicate that HIV-infected patients have nondeleterious autoantibodies recognizing cryptic epitopes exposed by pronase on T cells. (read more)

New Classification of Donation after Circulatory Death Donors Definitions and Terminology

In the face of a crisis in organ donation, the transplant community are increasingly utilising donation after circulatory death (DCD) donors. Over the last 10 years, with the increasing usage of DCD donors, we have seen the introduction in a number of new terms and definitions. We report the results of the 6th International Conference in Organ Donation held in Paris in 2013 and report a consensus agreement of an established expert European Working Group on the definitions and terminology regarding DCD donation, including refinement of the Maastricht definitions. This document forms part of a special series where recommendations are presented for uncontrolled and controlled DCD donation and organ specific guidelines for kidney, pancreas, liver and lung transplantation. An expert panel formed a consensus on definitions and terms aiming to establish consistent usage of terms in DCD donation (read more)

Deciphering IgM interference in IgG anti-HLA antibody detection with flow beads assays

In flow beads assays, the interference of IgM for IgG anti-HLA antibodies detection is not precisely understood. Using the screening flow beads assay for class I HLA antibodies, we analyzed the binding of two IgG mAbs, the anti-class I HLA W6/32 and an anti-beta-2-microglobulin, in the presence of an anti-beta-2-microglobulin IgM mAb. In neat serum, the IgM mAb impaired the detection of both IgG. In EDTA-treated serum, the interference was stronger for the anti-beta-2-microglobulin IgG than for W6/32, in agreement with the finding in surface plasmon resonance that this IgM competed with the anti-beta-2-microglobulin IgG but not with W6/32. The IgM interference was higher in neat than in EDTA-treated serum for both IgG mAbs. The IgM interference was also analyzed with class II single antigen flow beads and sera from two kidney recipients containing IgG and IgM donor specific antibodies. Anti-HLA IgG detection was partially corrected by EDTA, and restored by IgM inactivation with DTT, confirming the results observed with the mAbs. Therefore, three mechanisms can explain the IgM interference for IgG anti-HLA antibodies in flow beads assays: direct competition for antigen, steric hindrance and complement activation (read more)

Abdominal Wall Transplantation: A Sentinel Marker for Rejection

Abdominal wall (AW) transplantation (AWTX) has revolutionized difficult abdominal closure after intestinal transplantation (ITX). More importantly, the skin of the transplanted AW may serve as an immunological tool for differential diagnosis of bowel dysfunction post-transplant. Between 08/2008 and 10/2014, 29 small bowel transplantations were performed in 28 patients (16male;12female; 41±13years). Two groups were identified: The SOT (solid organ transplant)-group (n=15; 12ITX and 3MMVTX) and the SOT+AWTX-group (n=14; 12ITX and 2MMVTX), the latter included one ITX+AWTX retransplantation. Two doses of alemtuzumab were used for induction (30mg, 6 and 24hours post-reperfusion) and tacrolimus (trough levels 8–12ng/ml) was used for maintenance immunosuppression. Patient survival was similar in both groups (67%vs.61%). However, the SOT+AWTX-group showed faster post-transplant recovery, a better intestinal graft survival (79%vs.60%), a lower intestinal rejection rate (7%vs.27%) and a lower rate of misdiagnoses, where viral infection was mistaken and treated for rejection (14%vs.33%). The skin component of the AW may serve as an immune modulator and sentinel marker for immunological activity in the host. This can be a vital tool for timely prevention of intestinal graft rejection and more importantly avoidance of over-immunosuppression in cases of bowel dysfunction not related to graft rejection (read more).

The Role of Lymphoid Neogenesis in Allografts

De novo induction of organized lymphoid aggregates at non-lymphoid sites has been observed in many chronic inflammatory conditions where foreign antigens such as infectious agents, auto- or alloantigens, persist. The prevailing opinion in the field of transplantation is that lymphoid neogenesis within allografts is detrimental to the establishment of immune tolerance. These structures, commonly referred to as tertiary lymphoid organs (TLOs), are thought to contribute to graft rejection by generating and propagating local alloimmune responses. However, recent studies have shown that TLOs rich in regulatory Foxp3+ cells are present in long term accepting allografts. The notion that TLOs can contribute to the local downregulation of immune responses has been corroborated in other chronic inflammation models. These findings suggest that contrary to previous suggestions that the induction of TLOs in allografts is necessarily harmful, the induction of “tolerogenic” TLOs may prove advantageous. In this review, we discuss our current understanding of how TLOs are induced and how they regulate immune responses with a particular focus on alloimmunity (read more)

Renal Transplantation with Final Allocation Based on the Virtual Crossmatch

Solid phase immunoassays (SPI) are now routinely used to detect HLA antibodies. However the flow cytometric crossmatch (FCXM) remains the established method for assessing final donor-recipient compatibility. Since 2005 we have followed a protocol whereby the final allocation decision for renal transplantation is based on SPI (not the FCXM). Here we report long term graft outcomes for 508 consecutive kidney transplants using this protocol. All recipients were negative for donor specific antibody by SPI. Primary outcomes are graft survival and incidence of acute rejection within one year (AR<1yr) for FCXM+ (n=54) and FCXM- (n=454) recipients. Median follow up is 7.1 years. FCXM+ recipients were significantly different from FCXM- recipients for the following risk factors: living donor (24% vs 39%, p=0.03), duration of dialysis (31.0 months vs 13.5 months, p=0.008), retransplants (17% vs 7.3%, p=0.04), % sensitized (63% vs 19%, p=0.001) and PRA>80% (20% vs 4.8%, p=0.001). Despite these differences, 5 year actual graft survival rates are 87 and 84% respectively. AR<1yr occurred in 13% FCXM+ and 12% FCXM- recipients. Crossmatch status was not associated with graft outcomes in any univariate or multivariate model. Renal transplantation can be performed successfully, using SPI as the definitive test for donor-recipient compatibility (read more)